The instant invention is directed to a soluble and stabilized form in aqueous media of the envelope glycoprotein gp41 of HIV-1 suitable for inducing an immune response against a human immunodeficiency virus type 1 (HIV-1), pharmaceutical compositions comprising said gp41, a method of treatment against a human immunodeficiency virus, and/or HIV related diseases or disorders.
HIV-1 encodes a 160 kDa envelope glycoprotein (gp160) precursor, which is proteolytically cleaved into the exterior (gp120) and transmembrane (gp41) glycoproteins.
In the glycoprotein mature envelope, the gp120 glycoprotein remains associated with the gp41 ectodomain through a noncovalent interaction. The native HIV-1 envelope glycoproteins exist predominantly as trimers at the surface of the viral membrane, which consists of three gp120 and three gp41 subunits and are anchored in the viral or infected cell membrane by the gp41 transmembrane region.
It has been shown that the binding of gp120 to the CD4 receptor induces conformational changes that promote subsequent interaction with one of a number of chemokine receptors (CXCR4, CCR5 . . . ). These binding events trigger conformational changes in gp41. In particular, studies by X-ray crystallography and nuclear magnetic resonance indicate that the viral envelope glycoprotein gp41 exists in at least three conformations, a native conformation (spike), a prefusogenic metastable conformation which is converted to a thermostable fusogenic “three hairpin” conformation following a triggering event, such as binding of HIV-1 virus particle to the membrane of target cells.
So, the binding of gp120 to cellular coreceptors induces the gp41 conversion from a prefusogenic form to a fusogenic form.
The linear organization of the gp41 includes a fusion peptide, an ectodomain (a N-terminal coiled-coil, a disulfide-bonded loop region, and a C-terminal a-helical segment) and a transmembrane domain.
In the fusogenic six-helix bundle of the gp41, three N-terminal helices form a trimeric coiled-coil, and three C-terminal helices pack in the reverse direction into three hydrophobic grooves on the surface of the coiled-coil. This helical-hairpin structure corresponds to the fusion-active conformation of gp41. Because the transmembrane anchor and the fusion peptide of the gp41 ectodomain are embedded in the viral and target cell membranes, respectively, the formation of the fusogenic hairpin structure results in the colocalization of the two membranes and thus overcomes the energy barrier for membrane fusion.
The envelope glycoproteins of HIV-1 represent the only realistic viral target for vaccine-induced neutralizing antibody responses because they promote viral membrane fusion through receptor-mediated conformational change and they are expressed on the surface of both virions and infected cells. Monomeric HIV-1 gp120 and derivatives were initially considered to be principal vaccine candidates. However, HIV-1 gp120 is highly variable and has repeatedly proven to be an immunogen ineffective at eliciting neutralizing antibodies against clinical HIV-1 isolates. Few of the antibodies raised by gp120 monomers effectively bind assembled HIV-1 envelope glycoprotein trimers.
In contrast, gp41 is an extremely immunogenic glycoprotein, inducing antibodies in essentially all HIV-infected individuals.
The ectodomain of gp41 is the most conserved region of the HIV-1 envelope, membrane protein which otherwise exhibits considerable genetic diversity even among closely related isolates.
Furthermore, the gp41 performs a critical role in maintaining the conformation and infectivity of the HIV-1 virions.
The antibodies targeting the six-helix bundle (fusogenic form) and prehairpin (prefusogenic form) structures arrest fusion under certain conditions. Antibodies having access to prehairpin and six-helix bundles conformations of gp41 would be capable of inhibiting gp41-mediated fusion. Furthermore, the six-helix bundle is an extremely stable structure.
Those observations allow considering the gp41 six-helix, under a modified form or not, as an attractive target for drugs and vaccine development.
In U.S. Pat. No. 6,455,265, it was shown that some gp41 derivatives could be particularly efficient for obtaining vaccines for preventing the pathogenic effects related to a HIV retroviral infection, with the proviso that the corresponding polypeptides have epitopes having a modified antigenicity so as to obtain a differential immune response with respect to the viral envelope and some self-proteins.
More precisely, it was discovered that conserved and immunodominant regions of the retroviral envelope could be responsible for harmful autoimmune phenomena, particularly in the case of the gp41 retroviral envelope. It was observed that certain immunodominant regions of the gp41 exhibit three-dimensional structural analogies and/or cross-reactivities with certain regions of some proteins of the human immune system, and in particular the interleukin 2 (IL-2).
Accordingly, it was proposed in U.S. Pat. No. 6,455,265 modified polypeptides obtained by modifying the antigenicity of the concerned epitope of the envelope protein, in order to obtain a differential immune response with respect to the viral envelope protein and these proteins of the human immune system, in particular IL-2.
According to WO2005/01033, such modified polypeptides with at least one antigenic region of native gp41 protein of HIV-1 have been disclosed
Generally, synthetic gp41 can be produced in transfected baculovirus or mammalian cells but the yield is lower than in E. coli. Furthermore, the glycosylation in baculovirus or mammalian cells is different from the glycosylation of human cells and is not necessary for the immunogenicity of the protein. Gp41 is in fact very immunogenic without glycosylation.
However, full length or shorter recombinant HIV-1 ectodomain of gp41 produced in E. coli generally forms insoluble precipitates (aggregates of gp41 trimeric form) in aqueous media at neutral pH.
There is still a need to produce high levels of gp41 proteins that may be devoid of immunodominant region that trigger antibodies with no neutralizing activities but keeping important gp41 regions to focus the immune response on relevant epitopes that retain their overall immunogenic activity.
However there is still a need for a vaccine that allows for inducing a versatile immune response against HIV infection, and in particular HIV-type 1 infection. There is also a need for the development of non-clade B vaccines, such as, for example, clade C strains.
There is also a need for the development of a vaccine with broad inhibitory spectrum allowing for cross-clade inhibition.
There is a need for a vaccine allowing to induce an innate and/or a humoral and/or cellular immune response against HIV-1 infection.
There is a need for a vaccine allowing to induce an immune response against HIV infection at the mucosal surface level and/or at the blood level.
There is a need for a vaccine suitable for inducing mucosal IgA and/or antibodies and/or systemic IgA and/or IgG antibodies capable of interfering with HIV entry across the mucosa and early cell infection under the mucosa.
There is a need for a vaccine suitable for inhibiting or reducing HIV entry across mucosal tissues, e.g. vaginal mucosal tissues through various mechanisms such as transcytosis and ADCC (Antibody Depedent Cell Cytotoxicity).
It is an object of the invention to satisfy to all those above-mentioned needs.